The effectiveness of chemotherapy in GBM remains severely limited by the frequent development of resistance. Several factors contribute to this challenge, including the highly infiltrative nature of GBM, as well as its pronounced genetic and cellular heterogeneity [47]. Given the ability of CBD to reduce both cell proliferation and migration, its potential in combination with standard chemotherapeutics was investigated next. The aim was to assess whether co-administration could enhance antitumour efficacy while enabling dose reduction of conventional drugs, thereby minimizing toxicity and improving therapeutic outcomes. While in vitro cytotoxicity is commonly emphasized during early preformulation stages, the evaluation of drug–drug compatibility is often overlooked. To address this, DSC analysis was performed to evaluate the chemical compatibility between CBD and each of the selected chemotherapeutic agents. This compatibility assessment is a critical step in the context of co-delivery systems, where two or more drugs are encapsulated together within the same nanocarrier to ensure the same biodistribution upon administration. Unlike other combination therapies in which drugs may be administered separately and interact only transiently in the biological milieu, co-loaded formulations require that both compounds remain chemically stable in proximity, not only during administration and distribution, but also throughout manufacturing and storage. Ensuring such compatibility is essential to maintain the integrity and therapeutic performance of the formulation over its entire shelf-life and represents a foundational aspect of translational research.

TMZ, an alkylating agent approved by the FDA and EMA in 2005 and 2009, respectively, for the treatment of adult patients with newly diagnosed GBM, remains the first-line chemotherapy for GBM combined with surgical resection and radiotherapy [12,13]. Due to ongoing trials combining CBD: THC with TMZ (NCT03529448 and NCT05629702), TMZ was selected as the reference cytotoxic agent to be tested alongside CBD in this study.

At 24 and 48 h, the IC₅₀ values of TMZ were comparable between both glioma cell lines (Table 1). However, in the U-87 MG cell line, TMZ exhibited cytotoxic activity in a time-dependent manner (2.1 × 10³ μM and 1.3 × 10³ μM after 24 and 48 h, respectively), although in all cases IC₅₀ values were in the millimolar range.

In vitro analysis of the CBD and TMZ combination revealed a significantly enhanced reduction in cell viability in both U-87 MG and U-373 MG glioma cell lines, particularly when higher concentrations of CBD (25 µM) were combined with lower concentrations of TMZ (Figure 4a,b). This suggests a potential for dose reduction of TMZ, thereby minimizing associated toxicity. These findings are consistent with previous work by Nabissi et al. [37]. The effect was time-dependent and became more pronounced at 48 h. At 24 h, Bliss synergy scores indicated an additive interaction in U-87 MG cell line with a Bliss score of 3.98 ± 3.72 and a synergistic effect in U-373 MG with a Bliss score of 13.76 ± 4.78. At 48 h, Bliss scores rose to 9.66 ± 2.46 in U-87 MG, indicating a slightly additive synergistic effect, and to 20.20 ± 3.74 in U-373 MG, reflecting an enhanced synergistic effect (Table 2). The observed synergy likely stems from the complementary mechanisms of action of CBD and TMZ, reinforcing the potential of this combination to enhance therapeutic efficacy in GBM treatment while reducing TMZ-related toxicity.

In terms of physicochemical characterization, DSC analysis revealed a single exothermic peak for TMZ at 207.03 °C, consistent with its thermal degradation profile previously reported by Ahad et al. [48]. In the case of CBD, a well-defined endothermic peak was observed at 69.45 °C, corresponding to its melting point, in agreement with prior findings by Li et al. [49] and by Vlad et al. [50]. However, the DSC thermogram of the binary mixture (Figure 4c) revealed a splitting of the characteristic exothermic peak of TMZ into two distinct exothermic events in the presence of CBD. This suggests a possible interaction between the two compounds, potentially leading to the formation of multiple degradation products with different thermal characteristics. These results indicate a lack of physicochemical compatibility between CBD and TMZ under the tested conditions, which may pose challenges for their co-encapsulation within a single delivery system. Therefore, alternative combinations with more favourable compatibility profiles were explored to fully leverage the benefits of co-delivery systems.

Moving forward in the pursuit of effective therapeutic alternatives for GBM, BCNU, a chemotherapeutic agent formerly included in its gold-standard treatment as local therapy [51], was selected as a second candidate based on its DNA-alkylating mechanism, which closely resembles that of TMZ. Therefore, it was hypothesized that a similar synergistic effect might be achieved with BCNU while preserving drug compatibility. Additionally, the clinical use of BCNU is limited by its short plasma half-life and systemic toxicity, which, taken together, make BCNU a suitable candidate for benefiting from encapsulation within nanocarriers.

First, in vitro results demonstrated a more potent antiproliferative effect for BCNU compared to TMZ (Table 1). U-87 MG cells only exhibited greater sensitivity to BCNU than U-373 MG cells after 48 h, with IC₅₀ values of 1.7 × 10² µM and 2.7 × 10² µM, respectively. No time-dependency was evidenced in monotherapy with none of the glioma cell lines.

When combining CBD with BCNU in U-87 MG cells, two regions could be observed at both timepoints: whereas at lower concentrations of BCNU an additive effect was evidenced, at higher concentrations of BCNU an antagonistic effect was revealed. As a result of this antagonistic effect at higher BCNU concentrations, Bliss scores reached negative values of −2.25 ± 5.42 at 24 h and −19.10 ± 5.07 at 48 h (Figure 5a). In the case of U-373 MG cells, a time-dependent effect on the combination therapy was also displayed, with an antagonistic effect observed after 24 h, whereas at 48 h an additive effect emerged at higher concentrations of CBD. This time-dependency was supported by the Bliss score values that shifted from a strong antagonistic effect at 24 h (Bliss score: −24.37 ± 4.02) to an additive/slightly antagonistic interaction after 48 h (Bliss score: −9.89 ± 4.23) (Figure 5b).

The overall antagonistic interaction was further supported by DSC analysis, which revealed that, apart from the characteristic exothermic peak of BCNU at 137.55 °C [52], an additional exothermic peak emerged when combined with CBD at 174.67 °C. This thermal behaviour suggests that BCNU undergoes degradation in the presence of CBD (Figure 5c). Due to the lack of positive effects, this combination was not considered further in this study.

Given the failure of the combinations of CBD with TMZ and BCNU, the two standard chemotherapeutics used in GBM treatment, new combinations were explored with alternative chemotherapeutic agents that have not yet been considered in the clinical setting for GBM. The third alternative tested was DOX. Although the exact mechanism of action of DOX remains controversial, it is known to exert its cytotoxic effects through multiple pathways, including DNA intercalation and adduct formation, inhibition of topoisomerase II, generation of free radicals and oxidative stress, and disruption of cellular membranes [53]. On the one hand, the antitumour efficacy of DOX in GBM is limited by its poor BBB crossing properties, owing to P-glycoprotein (P-gp) efflux [54]. On the other hand, its clinical use is limited by its risk of cardiotoxicity [55]. The rationale for exploring the combination of CBD and DOX in GBM lies not only in their individual antineoplastic properties but also in preclinical evidence indicating that CBD may reduce DOX-induced cardiotoxicity when co-administered intraperitoneally in animal models [56].

As shown in Table 1, DOX exhibited a stronger antiproliferative effect than the previous alkylating agents, with comparable potency in both glioma cell lines. Notably, the antiproliferative effect of DOX followed a clear time-dependent pattern in both cell lines. For U-87 MG, the IC₅₀ decreased from 61 µM at 24 h to 3.5 × 10⁻¹ µM at 48 h, while for U-373 MG this value was reduced from 6.64 µM at 24 h to 8.9 × 10⁻¹ µM at 48 h.

In agreement with the time-dependency evidenced in DOX monotherapy, this effect was likewise present in combination therapy in both cell lines and particularly for the highest CBD concentrations (Figure 6a,b). This time-dependency was also supported by the Bliss score values in both cell lines (Table 2). The shift in Bliss score values was especially relevant for U-87 MG cells, where Bliss scores shifted from a strong antagonistic effect at 24 h (−17.03 ± 6.03) to an additive interaction after 48 h (−5.12 ± 3.63).

Furthermore, DSC analysis of the CBD + DOX combination revealed a thermal shift indicative of physicochemical incompatibility. The characteristic endothermic melting peak of DOX at 234.43 °C, previously reported by Gao et al. [57] and associated with its crystalline structure, was replaced by an exothermic event. This transition suggests a loss of crystallinity and potential degradation of DOX in the presence of CBD (Figure 6c). Overall, although CBD may offer cardioprotective effects, its combination with DOX did not improve therapeutic efficacy and could even be counterproductive, limiting its potential for future studies.

Finally, PTX, a microtubule-stabilizing agent known to induce mitotic arrest and cell death [58], was evaluated. On the one hand, the antitumour efficacy of PTX in GBM is critically hampered by its inability to reach effective concentrations in the central nervous system, largely due to its high affinity for the P-gp efflux transporter [59,60], which has led to its exclusion from standard GBM treatment protocols. Furthermore, taxanes, particularly PTX, are associated with a high incidence of peripheral neuropathy, affecting approximately 60–70% of patients undergoing chemotherapy [61,62]. However, preclinical studies have shown that cannabinoids confer neuroprotective effects against paclitaxel-induced peripheral neuropathy, preventing mechanical and thermal allodynia, preserving neurite outgrowth in sensory neurons, and reducing neuronal damage via mechanisms involving 5-HT_1A and CB₂ receptors. Therefore, studying the combination of CBD and PTX not only holds potential for enhancing the cytotoxicity of PTX against tumour cells but also for mitigating its neurotoxicity [63,64].

Among all compounds tested in this study, PTX showed the highest potency, reaching the lowest IC₅₀ values among all tested drugs (Table 1). In U-87 MG cells, the antiproliferative effect was time-dependent, with the IC₅₀ decreasing nearly 250-fold from 15 µM at 24 h to 5.9 × 10⁻² µM at 48 h. In U-373 MG cells, although PTX also exhibited strong antiproliferative activity, no time-dependent trend was observed.

The combination of CBD and PTX resulted in a synergistic interaction in both glioma cell lines, with enhanced cytotoxic effects particularly evident at higher concentrations of CBD and lower concentrations of PTX (Figure 7a,b). With regard to the Bliss scores, in the U-87 MG cell line, the combination therapy showed a synergistic effect at 24 h (Bliss score: 11.58 ± 5.49) that shifted into an additive effect after 48 h (Bliss score: 6.62 ± 5.49). For the U-373 MG cell line, an additive effect is maintained over the 48 h period. These findings suggest that the combined treatment could allow for a reduction in the effective dose of PTX, potentially minimizing its dose-limiting peripheral neuropathy while preserving antitumour efficacy.

Furthermore, the DSC analysis (Figure 7c) revealed that the characteristic thermal peaks of PTX—an endothermic peak at 223.68 °C, corresponding to its melting point and a secondary exothermic transition around 244.26 °C, corresponding to its degradation temperature [65], remained clearly identifiable in the thermogram of the binary mixture, indicating the absence of significant interactions between the two compounds. Taken together, the additive/synergistic antitumour activity observed in vitro and the confirmed thermal compatibility support the CBD + PTX combination as the most promising candidate. Consequently, subsequent studies focused on this combination to further explore its therapeutic potential in ovo.